Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro

BACKGROUND: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. METHODS: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. RESULTS: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. CONCLUSION: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action

Acute ischemic heart disease and interventional cardiology: a time for pause

BACKGROUND: A major change has occurred in the last few years in the therapeutic approach to patients presenting with all forms of acute coronary syndromes. Whether or not these patients present initially to tertiary cardiac care centers, they are now routinely referred for early coronary angiography and increasingly undergo percutaneous revascularization. This practice is driven primarily by the angiographic image and technical feasibility. Concomitantly, there has been a decline in expectant or ischemia-guided medical management based on specific clinical presentation, response to initial treatment, and results of noninvasive stratification. This 'tertiarization' of acute coronary care has been fuelled by the increasing sophistication of the cardiac armamentarium, the peer-reviewed publication of clinical studies purporting to show the superiority of invasive cardiac interventions, and predominantly supporting (non-peer-reviewed) editorials, newsletters, and opinion pieces. DISCUSSION: This review presents another perspective, based on a critical reexamination of the evidence. The topics addressed are: reperfusion treatment of ST-elevation myocardial infarction; the indications for invasive intervention following thrombolysis; the role of invasive management in non-ST-elevation myocardial infarction and unstable angina; and cost-effectiveness and real world considerations. A few cases encountered in recent practice in community and tertiary hospitals are presented for illustrative purposes The numerous and far-reaching scientific, economic, and philosophical implications that are a consequence of this marked change in clinical practice as well as healthcare, decisional and conflict of interest issues are explored. SUMMARY: The weight of evidence does not support the contemporary unfocused broad use of invasive interventional procedures across the spectrum of acute coronary clinical presentations. Excessive and unselective recourse to these procedures has deleterious implications for the organization of cardiac health care and undesirable economic, scientific and intellectual consequences. It is suggested that there is need for a new equilibrium based on more refined clinical risk stratification in the treatment of patients who present with acute coronary syndromes

Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter

Subcellular Antigen Location Influences T-Cell Activation during Acute Infection with Toxoplasma gondii

Effective control of the intracellular protozoan parasite Toxoplasma gondii depends on the activation of antigen-specific CD8+ T-cells that manage acute disease and prevent recrudescence during chronic infection. T-cell activation in turn, requires presentation of parasite antigens by MHC-I molecules on the surface of antigen presenting cells. CD8+ T-cell epitopes have been defined for several T. gondii proteins, but it is unclear how these antigens enter into the presentation pathway. We have exploited the well-characterized model antigen ovalbumin (OVA) to investigate the ability of parasite proteins to enter the MHC-I presentation pathway, by engineering recombinant expression in various organelles. CD8+ T-cell activation was assayed using ‘B3Z’ reporter cells in vitro, or adoptively-transferred OVA-specific ‘OT-I’ CD8+ T-cells in vivo. As expected, OVA secreted into the parasitophorous vacuole strongly stimulated antigen-presenting cells. Lower levels of activation were observed using glycophosphatidyl inositol (GPI) anchored OVA associated with (or shed from) the parasite surface. Little CD8+ T-cell activation was detected using parasites expressing intracellular OVA in the cytosol, mitochondrion, or inner membrane complex (IMC). These results indicate that effective presentation of parasite proteins to CD8+ T-cells is a consequence of active protein secretion by T. gondii and escape from the parasitophorous vacuole, rather than degradation of phagocytosed parasites or parasite products

Physiological aspects of the determination of comprehensive arterial inflows in the lower abdomen assessed by Doppler ultrasound

Non-invasive measurement of splanchnic hemodynamics has been utilized in the clinical setting for diagnosis of gastro-intestinal disease, and for determining reserve blood flow (BF) distribution. However, previous studies that measured BF in a "single vessel with small size volume", such as the superior mesenteric and coeliac arteries, were concerned solely with the target organ in the gastrointestinal area, and therefore evaluation of alterations in these single arterial BFs under various states was sometimes limited to "small blood volumes", even though there was a relatively large change in flow. BF in the lower abdomen (BFAb) is potentially a useful indicator of the influence of comprehensive BF redistribution in cardiovascular and hepato-gastrointestinal disease, in the postprandial period, and in relation to physical exercise. BFAb can be determined theoretically using Doppler ultrasound by subtracting BF in the bilateral proximal femoral arteries (FAs) from BF in the upper abdominal aorta (Ao) above the coeliac trunk. Prior to acceptance of this method of determining a true BFAb value, it is necessary to obtain validated normal physiological data that represent the hemodynamic relationship between the three arteries. In determining BFAb, relative reliability was acceptably high (range in intra-class correlation coefficient: 0.85-0.97) for three arterial hemodynamic parameters (blood velocity, vessel diameter, and BF) in three repeated measurements obtained over three different days. Bland-Altman analysis of the three repeated measurements revealed that day-to-day physiological variation (potentially including measurement error) was within the acceptable minimum range (95% of confidence interval), calculated as the difference in hemodynamics between two measurements. Mean BF (ml/min) was 2951 ± 767 in Ao, 316 ± 97 in left FA, 313 ± 83 in right FA, and 2323 ± 703 in BFAb, which is in agreement with a previous study that measured the sum of BF in the major part of the coeliac, mesenteric, and renal arteries. This review presents the methodological concept that underlies BFAb, and aspects of its day-to-day relative reliability in terms of the hemodynamics of the three target arteries, relationship with body surface area, respiratory effects, and potential clinical usefulness and application, in relation to data previously reported in original dedicated research

Exercise and the Prevention of Oesophageal Cancer (EPOC) study protocol: a randomized controlled trial of exercise versus stretching in males with Barrett's oesophagus

<p>Abstract</p> <p>Background</p> <p>Chronic gastro-oesophageal reflux disease and excessive body fat are considered principal causes of Barrett's oesophagus (a metaplastic change in the cells lining the oesophagus) and its neoplastic progression, oesophageal adenocarcinoma. Metabolic disturbances including altered levels of obesity-related cytokines, chronic inflammation and insulin resistance have also been associated with oesophageal cancer development, especially in males. Physical activity may have the potential to abrogate metabolic disturbances in males with Barrett's oesophagus and elicit beneficial reductions in body fat and gastro-oesophageal reflux symptoms. Thus, exercise may be an effective intervention in reducing oesophageal adenocarcinoma risk. However, to date this hypothesis remains untested.</p> <p>The 'Exercise and the Prevention of Oesophageal Cancer Study' will determine whether 24 weeks of exercise training will lead to alterations in risk factors or biomarkers for oesophageal adenocarcinoma in males with Barrett's oesophagus. Our primary outcomes are serum concentrations of leptin, adiponectin, tumour necrosis factor-alpha, C-reactive protein and interleukin-6 as well as insulin resistance. Body composition, gastro-oesophageal reflux disease symptoms, cardiovascular fitness and muscular strength will also be assessed as secondary outcomes.</p> <p>Methods/Design</p> <p>A randomized controlled trial of 80 overweight or obese, inactive males with Barrett's oesophagus will be conducted in Brisbane, Australia. Participants will be randomized to an intervention arm (60 minutes of moderate-intensity aerobic and resistance training, five days per week) or a control arm (45 minutes of stretching, five days per week) for 24 weeks. Primary and secondary endpoints will be measured at baseline (week 0), midpoint (week 12) and at the end of the intervention (week 24).</p> <p>Discussion</p> <p>Due to the increasing incidence and very high mortality associated with oesophageal adenocarcinoma, interventions effective in preventing the progression of Barrett's oesophagus are urgently needed. We propose that exercise may be successful in reducing oesophageal adenocarcinoma risk. This primary prevention trial will also provide information on whether the protective association between physical activity and cancer is causal.</p> <p>Trial Registration</p> <p>ACTRN12609000401257</p

Vitamin D exposure and Risk of Breast Cancer: a meta-analysis

The relationship between vitamin D and breast cancer is still controversial. The present meta-analysis examines the effects of the 25(OH)D, 1,25(OH)2D and vitamin D intake on breast cancer risk. For this purpose, a PubMed, Scopus and Web of Science-databases search was conducted including all papers published with the keywords "breast cancer" and "vitamin D" with at least one reported relative risk (RR) or odds ratio (OR). In total sixty eight studies published between 1998 and 2018 were analyzed. Information about type of study, hormonal receptors and menopausal status was retrieved. Pooled OR or RR were estimated by weighting individual OR/RR by the inverse of their variance Our study showed a protective effect between 25 (OH) D and breast cancer in both cohort studies (RR?=?0.85, 95%CI:0.74-0.98) and case-control studies (OR?=?0.65, 95%CI: 0.56-0.76). However, analyzing by menopausal status, the protective vitamin D - breast cancer association persisted only in the premenopausal group (OR?=?0.67, 95%CI: 0.49-0.92) when restricting the analysis to nested case-control studies. No significant association was found for vitamin D intake or 1,25(OH)2D. CONCLUSION: This systematic review suggests a protective relationship between circulating vitamin D (measured as 25(OH) D) and breast cancer development in premenopausal women